The present invention relates to an apparatus and method for imaging infrared-spectrum radiation that is emitted during cellular and molecular events, such as chemical reactions.
In assay screening, a large number of cellular events (e.g., calcium flux, etc.), physiological events and/or molecular events (e.g., chemical reactions, etc.) are monitored and analyzed. These events, hereinafter referred to as xe2x80x9ctarget events,xe2x80x9d are usually carried out in parallel in an array of deposits on specimen plates. The specimen plates are typically glass or plastic slides, or multi-well (e.g., micro-titer) plates.
Due to the large number of events taking place on the plates, time-consuming methods that directly examine each deposit (e.g., microscopic examination, etc.) are unsuitable for data acquisition. Rather, a xe2x80x9csnap shotxe2x80x9d of the whole plate is advantageously taken via imaging systems.
Area visible-spectrum imaging techniques, such as fluorescence imaging and luminescence imaging, can be used for data acquisition. In fluorescence imaging, when a target event occurs, a detection reagent emits light (i.e., fluoresces) when excited by an appropriate excitation source, e.g., ultraviolet light, etc. The detection reagent is chosen for its ability to interact (e.g., bind, etc.) with a target or to respond to a specific stimulus that is present only if the target event occurs. The emitted light, which provides quantitative information about the event, is captured and converted to electrical signals using, for example, a charge coupled device (xe2x80x9cCCDxe2x80x9d). The CCD comprises an array of thousands of sensor cells that are capable of receiving radiation from multiple wells at the same time. The signals are analyzed, via suitable software, to recover information concerning the target events.
Area fluorescent imaging devices are very complex and, hence, very expensive (c.a., $100,000 to $400,000). These imaging devices typically include an excitation light source, complicated optics, filters, a CCD, in some cases a cooler for the CCD, a control unit, software, positioners, and other elements. See, for example, the fluorescence imagers (FLIPR systems) available from Molecular Devices Corporation (www.moldev.com).
Luminescent imaging (chemi- or bio-) is similar to fluorescence imaging, except that excitation radiation is not required; the target event itself emits light. But many of the luminescent reactions have such low intensity emissions that a highly optimized imaging system, including the most sensitive form of cooled CCD camera and very efficient lenses, are required.
In addition to the high cost of these imaging devices, fluorescence imaging is complicated by the requirement of having a suitable detector reagent. While specific detector reagents have been developed for various applications, there are no universally applicable reagents. See, for example, Molecular Probes, Inc. (www.probes.com).
Consequently, a less costly and less complicated alternative to visible spectrum (i.e., fluorescence and luminescence) imaging is desirable. One possible alternative is thermal or infrared (IR) imaging.
All chemical reactions and physiological processes are accompanied by a change in energy; in other words, heat is generated or absorbed. Useful information can be obtained by monitoring/measuring such thermal changes.
Several methods are available for measuring thermal changes, including, for example, calorimetry and infrared thermography. Regarding the latter technique, published PCT application WO 99/60630 discloses a method of using infrared thermography to monitor physiological and molecular events.
According to that application, a high-resolution infrared imaging system is used to monitor heat output. FIG. 1 depicts a simplified schematic of the imaging system disclosed in WO 99/60630. Imaging system 100 comprises an infrared camera 102, including optics 104, that is spaced apart (i.e., the lens has a 6 centimeter focal length) from target 106 (e.g., a multi-well plate containing reagents, cellular or non-cellular material, a living animal, etc.). Target 106 is contained within isothermal chamber 108 that reduces temperature variations. The infrared camera monitors radiated heat production from target 106 and images are recorded by central processing unit 110 for data capture and analysis.
Infrared camera 102 advantageously provides a thermal image of the entire scene. That is, if a multi-well plate with its two-dimensional array of wells is being monitored, a thermal image of each well is preferably obtained. To do this, camera 102 must either (1) incorporate a scanning mechanism that sequentially focuses radiation from each well (or groups of wells) onto the detector or (2) use a focal plane array or xe2x80x9cstaringxe2x80x9d array.
Infrared cameras that incorporate scanning mechanisms are quite complicated. Scanning mechanisms typically comprise multiple movable reflective surfaces, a drive system to move the reflective surfaces and several lenses to focus incoming IR radiation onto the reflective surfaces. Furthermore, infrared cameras having scanning mechanisms cannot support ratiometric or comparative analysis of target events in each well, since this requires simultaneous image acquisition across all locations on the specimen plate. A focal plane array (xe2x80x9cFPAxe2x80x9d) 212, depicted in FIG. 2, is a monolithic microelectronic device that incorporates thousands of sensing elements 214 that continuously receive IR radiation, capturing an image of the entire scene. FPA-based infrared cameras, such as camera 302 depicted in FIG. 3, do not require a scanning system. Rather, they include a single monolithic FPA detector 212 and optics 104. Consequently, FPA-based cameras are lighter, quieter, consume less power, are more reliable, more durable and have a lower parts count than scan-based cameras. Furthermore, FPA-based cameras support ratiometric analysis. Ray tracing 318 depicts the relatively straight optical path of IR radiation from a target to detector 212 in FPA-based camera 302.
Regardless of whether the imaging system disclosed in WO 99/60630 uses a scanning system or an FPA-based camera as described above, the imaging system has certain characteristic drawbacks, which are discussed in conjunction with FIG. 4.
FIG. 4 depicts a simplified representation of the path 420 of IR radiation from target 106, through optics 104 to FPA 212. As shown in FIG. 4, the IR radiation must traverse medium 422 (e.g., air, etc.) between target 106 and optics 104, pass through optics 104 and then travel through medium 424 between optics 104 and FPA 212. The passage of IR radiation through media 422 and 424, and optics 104 attenuates the IR radiation, thereby compromising the sensitivity and resolution of the detector. Furthermore, passage of IR radiation through optics 104 introduces parallax-related aberrations.
The art would therefore benefit from infrared spectrum-imaging systems that avoid the complexity, performance deficits (e.g., reduced signal-to-noise ratio), expense and other drawbacks of prior art infrared-system imaging systems.
The present invention is an imaging apparatus and a method for imaging by which target events are monitored. Some infrared-spectrum imaging systems in accordance with the illustrative embodiment of the present invention comprise a two-dimensional detector array (hereinafter xe2x80x9cdetectorxe2x80x9d), which receives infrared radiation emitted from a specimen plate where target events are occurring. The detector is electrically connected to processing electronics that are operable to analyze the image data.